Project Abstract The class of regulatory T cells that has gained the most attention is called CD4+CD25+ regulatory T cells. This class of regulatory T cells is generated in the thymus. Tregs leaving the thymus are a homogenous population of CD44loCD62LhiCCR7+ cells that preferentially migrate to secondary lymphoid organs. Consistent with their self-reactivity, upon entering the periphery, a subset of Tregs acquire an activated phenotype, upregulating expression of CD44 and other homing receptors that allow them to access non-lymphoid tissues. This proper distribution of Tregs is fundamental for their ability to maintain tolerance, suggesting a model whereby distinct Tregs subsets are specialized for functioning either within secondary lymphoid organs to inhibit T cell priming, or in specific non-lymphoid tissues and sites of inflammation to dampen effector cell responses. Expression of CD44 and CD62L can be used to define ?central? TR (cTregs) and ?effector? Tregs (eTregs) that differentially localize to lymphoid and non-lymphoid tissues respectively. Overall, the activation of eTregs leads to their expansion into highly suppressive, but short-lived cells well-suited to regulating inflammatory responses in non- lymphoid tissues. Sphingosine-1-phosphate (S1P) is a bioactive lipid with important functions in the immune system. The marked difference in S1P concentrations between the circulation and tissues constitutes the S1P gradient that drives the trafficking of various immune cells. Recently, S1P and S1P receptors have been specifically targeted as treatments for immune-mediated diseases, particularly the immunosuppressant FTY720 (Fingolimod Gilenya), an S1P analogue that has recently become the first oral therapy for relapsing Multiple Sclerosis. In MS patients, FTY720 effectively decreases relapse rates better than interferon Beta therapy; however, a subset of patients treated with FTY720 develop very severe relapses and even tumorfactive lesions. This finding suggests that S1P1-mediated signaling may be involved in some unknown immune regulation, in addition to its role in T trafficking. To address the specific role of S1P on Tregs, we have generated mice that lack S1P1 only in Tregs. Surprisingly, S1P1 Treg-deficient mice developed rapid and severe systemic autoimmunity, revealing a critical role of S1P for the function of Tregs. Interestingly, most Tregs found in S1P1 Treg-deficient mice are eTregs, which are more prone to die of apoptosis. The analysis of Tregs derived from MS patients treated with Gilenya or other oral drugs, such as Tecfidera, has revealed a dramatic change in Treg homeostasis and an increase in the number of eTregs only in patients treated with Gilenya. Based on our preliminary data, we hypothesized that S1P1-mediated signaling regulates the function of Tregs by inhibiting their differentiation into short-lived eTregs. In this study we propose to elucidate how S1P1-mediated signaling regulates the differentiation of eTregs.